New Approaches for Probing PARylation in living Cells

聚腺苷二磷酸核糖化（Poly（ADP-ribos）ylation，PARylation）是大多数真核生物中一种重要的翻译后修饰和信号转导事件。像DNA修复和转录这样的基本过程是由这种瞬时聚合物协调的，它与蛋白质结合。ADP-核糖基转移酶（ART）从NAD+到各种受体蛋白上构建复杂的ADP-核糖链。修饰的复杂性和缺乏合适的工具使得PAR化的分子研究具有挑战性。在我们成功的早期工作的基础上，已经导致了几个联合出版物，这个合作项目的主要目的是建立在高时空分辨率的活细胞中DNA光损伤诱导的蛋白质PARylation动力学的定量研究方法。我们的方法是基于新的合成NAD+为基础的工具和先进的光学显微镜技术的发展。基于初步结果，马克思小组将开发出通过细胞膜传递修饰的NAD+类似物的方法，而不会干扰细胞活力。设想了两种修饰的NAD-类似物，其将允许成像（通过用荧光染料修饰）和通过脱硫生物素修饰的NAD+-类似物的加工修饰的蛋白质的亲和富集。Zumbusch小组将进一步开发光学实验，以优化NIR微辐射，从而使特定类型DNA损伤的受控诱导成为可能。为此，我们将建立独立控制用于微照射的NIR激光的脉冲能量和平均功率的方法。与此同时，将研究应用合成NAD+为基础的工具和PAR形成的光学显微镜的实验方案，以最大限度地减少对细胞健康的影响。这些将通过DNA光损伤后所选蛋白质的PAR化动力学的活细胞显微镜进行评估，并为未来的应用铺平道路。

Poly(ADP-ribos)ylation (PARylation) is a major post-translational modification and signalling event in most eukaryotes. Fundamental processes like DNA repair and transcription are coordinated by this transient polymer and it’s binding to proteins. ADP-ribosyltransferases (ARTs) build complex ADP-ribose chains from NAD+ onto various acceptor proteins. The complexity of the modification and the absence of suitable tools render molecular studies of PARylation challenging. Building on our successful earlier work that already lead to several joint publications, the major aim of this collaborative project is to establish means for quantitative studies of DNA photodamage induced protein PARylation dynamics in live cells with high temporal and spatial resolution. Our approach is based on the development of novel synthetic NAD+-based tools and advanced optical microscopy techniques. Based on the preliminary results, the Marx group will develop means to deliver modified NAD+-analogues through the cell membrane that do not interfere with the cell viability. Two kinds of modified NAD-analogues are envisioned that will allow imaging (through modification with a fluorescent dye) and affinity enrichment of protein that are modified through processing of desthiobiotin-modified NAD+-analogues. The Zumbusch group will further develop optical experiments to optimize NIR microirradiation such that controlled induction of specific types of DNA damages becomes possible. To this end, we will establish means to independently control pulse energy and average power of the NIR laser used for microirradiation. At the same time, experimental protocols for the application of synthetic NAD+-based tools and optical microscopy of PAR formation with minimized effects on cell health will be investigated. These will be evaluated by live cell microscopy of PARylation dynamics of selected proteins after DNA photo-damage and path the way for future applications.